Hsp70-derived peptides and uses thereof in the diagnosis and treatment of autoimmune diseases

ABSTRACT

The invention relates to specific peptides derived from hsp70, and to pharmaceutical compositions comprising the same. The peptides and compositions of the invention are particularly suitable for the prevention or treatment of an autoimmune disease such as Type 1 Diabetes, Systemic Lupus Erithematosus, Multiple Sclerosis or Rheumatoid Arthritis. The invention further relates to a method for diagnosing the occurrence or incipience of an autoimmune disease in a patient by use of the peptides of the invention, by testing a blood or urine sample of a patient for the presence of antibodies or T-cells which are immunologically reactive to human hsp70. The invention also relates to a kit for the diagnosis of an autoimmune disease by testing for the presence of anti-hsp70 antibodies by aid of the peptides of the invention.

FIELD OF THE INVENTION

The present invention relates to methods of treatment and diagnosis ofautoimmune diseases. More specifically, the invention relates to hsp70peptides and their use in the diagnosis and treatment of autoimmunediseases.

BACKGROUND OF THE INVENTION

Type 1 Diabetes (Insulin dependent diabetes mellitus, IDDM) is a diseasecaused by autoimmune T-cells that attack the insulin-producing β cellsof the pancreatic islets [Bach, J. F. (1994) Endocrine Reviews15:516-542; Atkinson, M. A. and Maclaren, N. K. (1994) New Engl. J. Med.331:1428-1436; Honeyman, M. C. and Harrison, L. C. (1993) SpringerSemin. Immunol pathol. 14(3):253-274]. In humans and in the NOD micemodel system, in which the condition develops spontaneously, the diseaseappears to involve autoimmunity to a similar collective of antigensincluding proinsulin and insulin [Honeyman, M. C., and Harrison, L. C.(1993) id ibid.; Roep B. O. (1996) Diabetes 45:1147-1156], glutamic aciddecarboxylase (GAD) [Harrison, L. C. et al. (1991) Diabetes 40(9):1-128-1133; Naquet, P. et al. (1988) J. Immunol. 140:2569-2578], theisle T-cell antigen ICA69 [Atkinson, M. A. et al. (1992) Lancet339:458-459], and the insulin secretory-granule 38 kDa protein (38 kDa)[Atkinson, M. A. et al. (1994) J. Clin. Invest. 94:2125-2129; reviewedin: Atkinson, M. A. and Maclaren, N. K. (1994) id ibid.]. In addition,the 60 kDa heat-shock protein (hsp60) is one of the auto-antigens foundin the NOD mouse model.

It is interesting that Type 1 diabetes patients and NOD mice appear tomake T-cell responses to similar hsp60 peptides. The similarity intarget peptides may result from the similar peptide-binding motifs ofthe mouse I-Ag7 [Reizis, B. et al. (1996) Internation. Immunol. 9 (1):43-51] and the human DQ8 MHC molecules [Kwok, W. W. et.al. (1996) J.Immunol. 156:2171-7], both associated with susceptibility to IDDM.

Studies in the NOD mouse model indicate that Type 1 Diabetes is a T-cellmediated disease, wherein the cells involved in the pathogenesis of thedisease are Th1-type T-cells. It has been shown that NOD micespontaneously develop T-cells responsive to a hsp60 peptide, and theseT-cells can adoptively transfer diabetes or, when attenuated, canvaccinate mice against diabetes (T-cell vaccination, TCV) [Elias, D. etal. (1991) Proc. Natl. Acad. Sci. USA 88:3088-91]. Moreover, a single,subcutaneous administration of a hsp60 peptide either early, at 4-6weeks of age [Elias, D. et al. (1991) id ibid.], or very late in theautoimmune process, at 12-17 weeks, can arrest the disease [Elias, D. etal. (1994) Lancet 343:704-706; Elias, D. and Cohen, I. R. (1995)Diabetes 44:1132-1138]. The same hsp60 peptide was also found toinfluence toxin-induced diabetes. Mice of the C57BL/KsJ strain can beinduced to develop a type of autoimmune diabetes about 3 months afteradministration of a very low dose of the β-cell toxin streptozotocin[Elias, D. et al. (1994) Diabetes 43:992-998]. This form of diabetescould also be treated with the hsp60 peptide administered after thetoxic insulitis. In contrast to the hsp60 peptide treatment, treatmentof the mice with an immunogenic GAD peptide failed to arrest thedevelopment of diabetes [Elias, D. and Cohen, IR. (1996) Diabetes45:1168-1172]. Effective treatment of the diabetic process in mice withhsp60 peptides appears to involve a temporary burst of“anti-inflammatory” Th2-like reactivity that down-regulates pathogenicTh1-like reactivity to hsp60. This down-regulation induced by hsp60appears to spread to down-regulate the Th1-like responses to otherantigens targeted in Type 1 Diabetes [Elias, D. et al. (1997) Diabetes46:758-764].

Anti-hsp60 T-cells can also mediate insulitis and hyperglycemia [Roep,B. O. et al. (1996) Euro. J. Immunol. 26(6):1285-1289], and modulatingthe anti-hsp60 T-cell response can lead to the arrest of the autoimmunedestruction of β cells [Roep B. O. et al. (1991) Lancet 337:1439-1441;Elias, D. et al. (1991) id ibid.]. Recently, the inventors reportedproliferative responses to human hsp60 and its peptides in 25 newlydiagnosed Type 1 Diabetes adult patients amongst whom 92% testedpositive to hsp60 [Abulafia-Lapid, R. et al. (1999) J. Autoimmunity12:121-129].

Several studies have suggested that other heat shock proteins, likehsp70 and hsp90, may also have a role in the pathogenesis of autoimmunedisorders [Lindquist, S. (1988) Annu. Rev. Genet. 22:631-677; Polla, B.S. and Young, D. (1989) Immunol. Today 10:393-394; Feige, U. and vanEden, W. (1996) Infection, autoimmunity and autoimmune disease EXS77:359-373]. A role for these molecules in antigen presentation has beenreported [Kaufman, S. H. E. (1990) Immunol. Today 11:129-136; VanBuskirk, A. et al. (1989) J. Exp. Med. 170:1799-1809], as well as theassociation of the 8.5 kD hsp70-2 allele with diabetic haplotypes[Pugliese, A. et al. (1992) Diabetes 41:788-791]. Moreover,auto-antibodies against hsp70 and hsp90 have been found in patientssuffering from systemic lupus erythematosus, polymyositis [Minota, S.and Winfield, J. (1988) Arthritis Rheum. 31:S13; Minota, S. et al.(1988) J. Clin. Invest. 81:106-119] and multiple sclerosis [Salvetti M.et al. (1996) J. Neuroimmunol. 65(2):143-153]. Therefore, it has beenfurther suggested that hsp70 and hsp90 might also be target antigens inType 1 Diabetes [Minota, S. et al. (1988) id ibid.].

In view of these suggested roles for hsp70, the inventors investigatedwhether children newly diagnosed with Type 1 Diabetes present T-cellproliferative responses to human hsp70 and hsp90. The inventors foundthat in children who were newly diagnosed as having Type 1 diabetes,there was a T-cell proliferative response to hsp70 but not to hsp90. Itis important that this response was measured in newly diagnosedchildren, since the T-cell response is acute and destructive until theα-cell islets are destroyed, as demonstrated by the T-cell response tohsp60 protein, which declines at about 16 weeks after diagnosis[Abulafia-Lapid et al. (1999) id ibid.]. In addition, the inventorstested the specific response to hsp70 peptides, while mapping the majorhsp70 protein epitopes. Finally, the presence of sera antibodies tohsp60, hsp70, and hsp90 was tested.

In view of the interesting results obtained with respect to theanti-hsp70 response, the inventors have developed methods for thediagnosis and treatment of autoimmune diseases in general, and morespecifically to Type 1 Diabetes, utilizing for this purpose the hsp70peptides of the invention.

It is therefore an object of the present invention to provide novelhsp70 peptides. It is also an object of the invention to provide methodsof diagnosis and treatment of autoimmune diseases using the novel hsp70peptides of the invention.

These and other objects of the invention will become more apparent asthe description proceeds.

SUMMARY OF THE INVENTION

The present invention relates to hsp70 peptides and their use in thediagnosis and treatment of autoimmune diseases, preferably Type 1Diabetes, Systemic Lupus Erithematosus, Multiple Sclerosis or RheumatoidArthritis, more preferably Type 1 Diabetes.

As presently claimed, in a first aspect, the present invention relatesto a peptide selected from the group consisting of the peptides denotedby SEQ. ID. NO.1, SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID.NO.5, SEQ. ID. NO.6, SEQ. ID. NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ.ID. NO.10, SEQ. ID. NO.11, SEQ. ID. NO.12, SEQ. ID. NO. 13, SEQ. ID.NO.14, SEQ. ID. NO.15, SEQ. ID. NO.16, SEQ. ID. NO.17, SEQ. ID. NO.18,SEQ. ID. NO.19, SEQ. ID. NO.20, SEQ. ID. NO.21, SEQ. ID. NO.22, SEQ. ID.NO.23, SEQ. ID. NO.24, SEQ. ID. NO.25, SEQ. ID. NO.26, SEQ. ID. NO.27,SEQ. ID. NO.28, SEQ. ID. NO.29, SEQ. ID. NO.30, SEQ. ID. NO.31, SEQ. ID.NO.32, SEQ. ID. NO.33, SEQ. ID. NO.34, SEQ. ID. NO.35, SEQ. ID. NO.36,SEQ. ID. NO.37, SEQ. ID. NO.38, SEQ. ID. NO.39, SEQ. ID. NO.40, SEQ. ID.NO.41, SEQ. ID. NO.42 and SEQ. ID. NO.43, and salts, analogues andfunctional derivatives thereof. Preferably, the peptide of the inventionis selected from the group consisting of the peptides SEQ. ID. NO.1,SEQ. ID. NO.12, SEQ. ID. NO.15, SEQ. ID. NO.16, SEQ. ID. NO.19, SEQ. ID.NO.27, SEQ. ID. NO.29, SEQ. ID. NO.34 and SEQ. ID. NO.35. Morepreferably, the peptide of the invention is selected from the groupconsisting of the peptides denoted by SEQ. ID. NO.1, SEQ. ID. NO.27 andSEQ. ID. NO.35.

Functional derivatives of the peptide of the invention consist ofchemical modifications to amino acid side chains and/or the carboxyland/or amino moieties of said peptides.

In a second aspect, the present invention relates to a pharmaceuticalcomposition comprising at least one peptide of the invention, andoptionally comprising a pharmaceutically acceptable carrier.

In a specific embodiment, the pharmaceutical composition of theinvention is for use in the prevention or treatment of an autoimmunedisease, preferably Type 1 Diabetes, Systemic Lupus Erithematosus,Multiple Sclerosis or Rheumatoid Arthritis, more preferably Type 1Diabetes.

In a third aspect, the present invention relates to a method fordiagnosing the occurrence or incipience of an immune disease in apatient, utilizing a peptide as defined in the first aspect of theinvention. Preferably, the immune disease is Type 1 Diabetes, SystemicLupus Erithematosus, Multiple Sclerosis or Rheumatoid Arthritis. Morepreferably, the immune disease is Type 1 Diabetes.

In one embodiment, the method of the invention comprises testing a bloodor urine sample of said patient for the presence of antibodies orT-cells which are immunologically reactive to human hsp70. Said methodinvolves contacting said sample with a peptide of the invention anddetecting an immunoreaction between said sample and said peptide,wherein the presence of such immunoreaction indicates the presence ofanti-hsp70 antibodies or of a T-cell which immunoreacts with hsp70,indicating an increased probability of the presence or incipience of anautoimmune disease.

In a specific embodiment, the presence of anti-hsp70 antibodies isrevealed by an immunoreaction detected by radioimmunoassay and/or by anELISA test or any other test that might detect the said anti-hsp70.

In a further specific embodiment, the method to test for the presence ofsaid T-cell which immunoreacts with hsp70 comprises the steps of:

-   (a) preparing a mononuclear cell fraction containing T-cells from a    blood sample obtained from said patient;-   (b) adding to said mononuclear cell fraction an antigen selected    from the peptides defined in the invention;-   (c) incubating said cell fraction in the presence of said antigen    for a suitable period of time and under suitable culture conditions;-   (d) adding a labeled nucleotide to the incubated cell culture of (c)    at a suitable time before the end of said incubation period to    provide for the incorporation of said labeled nucleotide into the    DNA of proliferating T-cells; and-   (e) determining by suitable means the amount of proliferating    T-cells by analysis of the amount of labeled nucleotide incorporated    into said T-cells.

In a further aspect, the invention relates to a kit for the diagnosis ofan autoimmune disease. Preferably the kit is for the diagnosis of Type 1Diabetes, Systemic Lupus Erithematosus, Multiple Sclerosis or RheumatoidArthritis. More preferably, said kit is for the diagnosis of Type 1Diabetes.

In one embodiment, said diagnosis is achieved by testing for thepresence of anti-hsp70 antibodies, wherein said kit comprises thefollowing components:

-   (a) at least one antigen selected from peptides of the invention;    and-   (b) a tagged antibody capable of recognizing the non-variable region    of said anti-hsp70 antibodies.

In another embodiment, said diagnosis is achieved by testing for thepresence of a T-cell which immunoreacts with hsp70, wherein said kitcomprises the following components:

-   (a) at least one antigen selected from the peptides of the    invention;-   (b) a suitable medium for culture of lymphocytes (T-cells); and-   (c) a labeled nucleotide for a T-cell proliferation test.

In a last aspect, the invention relates to a method of modulating animmune response, and arresting the autoimmune process, in a patient inneed of such treatment, wherein said method comprises administering tosaid patient, at least once, a peptide selected from the peptides of theinvention in a medically effective amount. Said method shall help thebody to stop the auto-immune process.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be more clearly understood from the detaileddescription of the preferred embodiments and from the attached figuresin which:

FIG. 1: T-cell responses of 25 Type 1 Diabetes children to the intacthsp60 and hsp70 molecules

T-cells were activated with 2-5 μg/ml hsp60 and 2-5 μg/ml hsp70. Theproliferative responses are presented in arbitrary S.I. (stimulationindex) units. A T-cell proliferation of S.I.≧2 was considered positive,and this cut off is marked by a horizontal dashed line. Abbreviations:pat., patients.

FIG. 2A-B: Epitope mapping of hsp70 protein

Seven Type 1 Diabetes patients (P3, P16, P18, P20, P21, P23 and P24)were tested for responsiveness to multiple hsp70 epitopes thatencompassed the entire human hsp70 molecule. T-cells isolated from theseven representative patients were assayed for proliferative responsesto the 43 overlapping hsp70 peptides (detailed in Table 2). T-cells wereactivated with 5-20 μg/ml of each peptide. A T-cell proliferation ofS.I.≧2 was considered positive.

FIG. 2A: Responses to peptides p1-p22.

FIG. 2B: Responses to peptides p23-p43.

Abbreviations: pat., patients; pept., peptides.

FIG. 3: Measurement of sera auto-antibodies to hsp60, hsp70 and hsp90.

This graph represents the levels of IgG antibodies to hsp60, hsp70 andhsp90 in the sera of 21 patients with Type 1 Diabetes (Table 1A) incomparison with the sera of 10 normoglycemic children. The level ofantibodies was considered positive when it was greater than the mean ofthe values of antibody levels obtained from the control group(normoglycemic children) plus two standard deviations. The mean of thevalues from the control group plus two standard deviations was thusconsidered as the cut-off level. Abbreviations: pos., positives; antig.,antigen; Diab. I, Type 1 Diabetes; healt., healthy; Tet. Tox., TetanusToxoid.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarification, the following terms are defined herein:

-   -   hsp: heat-shock protein.    -   IDDM: Insulin-dependent Diabetes Mellitus, recently denominated        Type 1 Diabetes.    -   NIDDM: Non-insulin Dependent Diabetes Mellitus, recently        denominated Type 2 Diabetes.    -   NOD mice: Non-Obese Diabetes Mice. A mouse model that develops a        spontaneous form of diabetes, considered a good model for Type 1        Diabetes. Female NOD mice develop insulitis at around 4 weeks of        age and hyperglycemia starts at about 14-17 weeks. By 35-40        weeks almost all female NOD mice have developed severe diabetes        and most die in the absence of insulin treatment.    -   PBMC: peripheral blood mononuclear cells.    -   S.I.: stimulation index, this index is calculated by dividing        the response (in cpm counts) by the cpm counts obtained in the        background (which is set by the counts given by T cells in        culture in the absence of the antigen).    -   TT: tetanus toxoid.

The inventors have found that a significant proportion of recentlydiagnosed Type 1 Diabetes children manifest T-cell proliferativeactivity to human hsp60 and hsp70 proteins, but not to hsp90. Mostimportantly, comparing the T-cell response to each one of hsp60, hsp70and hsp90, (referred to in the Examples as the Stimulation Index, S.I.),the inventors observed that the response to hsp70 was the highest inType 1 Diabetes patients (Example 1, Table 1A).

Curiously, subjects with Type 2 Diabetes did not show higher response toeither hsp60 or hsp70 (Example 1, Table 1C), than did healthy subjects(Example 1, Table 1B). Therefore, hsp70 may be a member of thecollective of self-antigens to which there is enhanced T-cell reactivityin Type 1 Diabetes, but not in Type 2 Diabetes [Roep, B. (1996) idibid.].

The present invention thus relates to methods of treatment and diagnosisof an autoimmune disease. More specifically, the invention relates tohsp70 peptides and their use in the diagnosis and treatment of saidautoimmune disease. Preferably, the autoimmune disease to be diagnosedor treated is Type 1 Diabetes, Systemic Lupus Erithematosus, MultipleSclerosis or Rheumatoid Arthritis. More preferably, said autoimmunedisease is Type 1 Diabetes.

In a first aspect, the present invention relates to hsp70 overlappingpeptides, selected from the group consisting of peptides denoted by anyone of SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4, SEQ.ID.NO.5,SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.10,SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15,SEQ.ID.NO.16, SEQ.ID.NO.17, SEQ.ID.NO.18, SEQ.ID.NO.19, SEQ.ID.NO.20,SEQ.ID.NO.21, SEQ.ID.NO.22, SEQ.ID.NO.23, SEQ.ID.NO.24, SEQ.ID.NO.25,SEQ.ID.NO.26, SEQ.ID.NO.27, SEQ.ID.NO.28, SEQ.ID.NO.29, SEQ.ID.NO.30,SEQ.ID.NO.31, SEQ.ID.NO.32, SEQ.ID.NO.33, SEQ.ID.NO.34, SEQ.ID.NO.35,SEQ.ID.NO.36, SEQ.ID.NO.37, SEQ.ID.NO.38, SEQ.ID.NO.39, SEQ.ID.NO.40,SEQ.ID.NO.41, SEQ.ID.NO.42, SEQ.ID.NO.43. Preferably, the peptides ofthe invention are selected from the group consisting of peptides denotedby any one of SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.15, SEQ.ID.NO.16,SEQ.ID.NO.19, SEQ.ID.NO.27, SEQ.ID.NO.29, SEQ.ID.NO.34 and SEQ.ID.NO.35.More preferably, the peptides of the invention are the peptides denotedby SEQ.ID.NO.1, SEQ.ID.NO.27 and SEQ.ID.NO.35.

The peptides of the invention may be used in free form or as salt, e.g.,as metal salt, including sodium, potassium, lithium or calcium salt, oras a salt with an organic base, or as a salt with a mineral acid,including sulfuric acid, hydrochloric acid or phosphoric acid, or withan organic acid e.g., acetic acid or maleic acid. Generally, anypharmaceutically acceptable salt of the peptide of the invention may beused, as long as the biological activity of the peptide with respect todiabetes is maintained.

Functional derivatives consist of chemical modifications to amino acidside chains and/or the carboxyl and/or amino moieties of said peptides.Modifications can also include backbone modifications, like insertions,deletions or replacement of any one of the amino acids of said peptides.

It is to be understood by all of skill in the art that suitable analogsof these new peptides may be readily synthesized by now-standard peptidesynthesis methods and apparatus. The only limitation on such analogs isthat they have essentially the same biological activity of the hsp70peptides with respect to diabetes. All such analogs will essentially bebased on the new peptides as regards their amino acid sequence but willhave one or more amino acid residues deleted, substituted or added. Whenamino acid residues are substituted, such substitutions which areenvisaged are those which do not significantly alter the structure orbiological activity of the peptide, for example basic amino acids willbe replaced with other basic amino acids, acidic ones with acidic onesand neutral ones with neutral ones. The overall length of the analogpeptide can be between about 9 to 35 amino acids. Preferably, when aminoacid residues are deleted, the same above restraints are applied asregards obtaining the aforesaid biologically active peptides, and alsowherein such deletion analogs will still have between about 17 to about23 amino acid residues (deletion analogs will usually be no less thanabout 13 amino acid residues in length). Further preferably, when aminoacid residues are added, the aforesaid restraints concerning biologicalactivity are applied and such addition analogs will usually still havebetween about 17 to about 23 amino acids (addition analogs will usuallybe up to about 30 amino acids in length).

In a second aspect, the invention relates to a pharmaceuticalcomposition comprising a peptide selected from the group consisting ofpeptides denoted by any one of SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.15,SEQ.ID.NO.16, SEQ.ID.NO.19, SEQ.ID.NO.27, SEQ.ID.NO.29, SEQ.ID.NO.34 andSEQ.ID.NO.35. Preferably, the pharmaceutical composition of theinvention comprises a peptide selected from the group consisting ofSEQ.ID.NO.1, SEQ.ID.NO.27 and SEQ.ID.NO.35. The pharmaceuticalcompositions of the invention may also comprise a mixture of at leasttwo of the peptides denoted by any one of SEQ.ID.NO.1, SEQ.ID.NO.12,SEQ.ID.NO.15, SEQ.ID.NO.16, SEQ.ID.NO.19, SEQ.ID.NO.27, SEQ.ID.NO.29,SEQ.ID.NO.34 and SEQ.ID.NO.35. Optionally, the pharmaceuticalcomposition also comprises pharmaceutically acceptable carrier, additiveand/or diluent.

The peptides of the invention may be used as such or in the form of acomposition. A composition will generally contain salts, preferably inphysiological concentration, such as PBS (phosphate-buffered saline), orsodium chloride (0.9% w/v), and a buffering agent, such as phosphatebuffer in the above PBS. The preparation of pharmaceutical compositionsis well known in the art, see e.g., U.S. Pat. Nos. 5,736,519, 5,733,877,5,554,378, 5,439,688, 5,418,219, 5,354,900, 5,298,246, 5,164,372,4,900,549, 4,755,383, 4,639,435, 4,457,917, and 4,064,236. The peptideof the present invention, or a pharmacologically acceptable salt thereofis preferably mixed with an excipient, carrier, diluent, and optionally,a preservative or the like pharmacologically acceptable vehicles asknown in the art, see e.g., the above US patents. Examples of excipientsinclude glucose, mannitol, inositol, sucrose, lactose, fructose, starch,corn starch, microcrystalline cellulose, hydroxypropylcellulose,hydroxypropylmethyl-cellulose, polyvinyl-pyrrolidone and the like.Optionally, a thickener may be added, such as a natural gum, a cellulosederivative, an acrylic or vinyl polymer, or the like.

The pharmaceutical composition is provided in solid, liquid orsemi-solid form. A solid preparation may be prepared by blending theabove components to provide a powdery composition. Alternatively, thepharmaceutical composition is provided as lyophilized preparation. Theliquid preparation is provided preferably as aqueous solution, aqueoussuspension, oil suspension or microcapsule composition. A semi-solidcomposition is provided preferably as hydrous or oily gel or ointment.

A solid composition may be prepared by mixing an excipient with asolution of the peptide of the invention, gradually adding a smallquantity of water, and kneading the mixture. After drying, preferably invacuum, the mixture is pulverized. A liquid composition may be preparedby dissolving, suspending or emulsifying the peptide of the invention inwater, a buffer solution or the like. An oil suspension may be preparedby suspending or emulsifying the peptide of the invention or protein inan oleaginous base, such as sesame oil, olive oil, corn oil, soybeanoil, cottonseed oil, peanut oil, lanolin, petroleum jelly, paraffin,Isopar, silicone oil, fatty acids of 6 to 30 carbon atoms or thecorresponding glycerol or alcohol esters. Buffers include Sorensenbuffer (Ergeb. Physiol., 12, 393 1912), Clark-Lubs buffer (J. Bact., 2,(1), 109 and 191, 1917), Macllvaine buffer (J. Biol. Chem., 49, 183,1921), Michaelis buffer (Die Wasserstoffinonenkonzentration, p. 186,1914), and Kolthoff buffer (Biochem. Z., 179, 410, 1926).

A composition may be prepared as a hydrous gel, e.g. for transnasaladministration. A hydrous gel base is dissolved or dispersed in aqueoussolution containing a buffer, and the peptide of the invention, and thesolution warmed or cooled to give a stable gel.

Preferably, the peptide of the invention is administered throughintravenous, intramuscular or subcutaneous administration. Oraladministration is expected to be less effective, because the peptide maybe digested before being taken up. Of course, this consideration mayapply less to a peptide of the invention which is modified, e.g., bybeing cyclic peptide, by containing non-naturally occurring amino acids,such as D-amino acids, or other modification which enhance theresistance of the peptide to biodegradation. Decomposition in thedigestive tract may be lessened by use of certain compositions, forinstance, by confining the peptide of the invention in microcapsulessuch as liposomes. The pharmaceutical composition of the invention mayalso be administered to other mucous membranes. The pharmaceuticalcomposition is then provided in the form of a suppository, nasal sprayor sublingual tablet. The dosage of the peptide of the invention maydepend upon the condition to be treated, the patient's age, bodyweight,and the route of administration, and will be determined by the attendingphysician.

In another embodiment, the peptide of the invention may be provided in apharmaceutical composition comprising a biodegradable polymer selectedfrom poly-1,4-butylene succinate, poly-2,3-butylene succinate,poly-1,4-butylene fumarate and poly-2,3-butylene succinate,incorporating the peptide of the invention as the pamoate, tannate,stearate or palmitate thereof. Such compositions are described e.g., inU.S. Pat. No. 5,439,688.

In another embodiment, a composition of the invention is a fat emulsion.The fat emulsion may be prepared by adding to a fat or oil about 0.1-2.4w/w of emulsifier such as a phospholipid, an emulsifying aid, astabilizer, mixing mechanically, aided by heating and/or removingsolvents, adding water and isotonic agent, and optionally, adjustingadding the pH agent, isotonic agent. The mixture is then homogenized.Preferably, such fat emulsions contain an electric charge adjustingagent, such as acidic phospholipids, fatty acids, bilic acids, and saltsthereof. Acidic phospholipids include phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. Bilicacids include deoxycholic acid, and taurocholic acid. The preparation ofsuch pharmaceutical compositions is described in U.S. Pat. No.5,733,877.

The inventors have mapped the responses of patients to a series ofoverlapping hsp70 peptides. FIG. 2 shows that hsp70 peptides p1, p12,p15, p16, p19, p27, p29, p34 and p35 triggered a T-cell response in theType 1 Diabetes patients tested.

The peptides of the invention contain (with the exception of p43) 20amino acids. It is known that the immunogenic motif that is recognizedby the antigen-presenting cell (APC), which display the HLA class II, isonly 9 amino acids long.

The auto-reactive T-cells involved in the autoimmune process areactivated by APCs. The APC processes the self-antigen and presents theimmuno-dominant peptide on the MHC (Major Histocompatibility Complex, inhuman known as HLA), which then becomes available for recognition byantigen-specific T-cells. Interestingly, certain HIA molecules have beenshown to be associated with auto-immunity due to the presentation ofdisease-associated peptides. For example, the HLA-DQB1*0302 has beendescribed as associated with Type 1 Diabetes, for preferentially bindingto GAD (glutamic acid decarboxylase), a protein that has been associatedwith the disease [W. W. et al. (1996) id ibid.]. Therefore, it remainsto be determined which HLA haplotypes are expressed by the Type 1Diabetes patients studied in the present invention, to understand howthese haplotypes correlate with the specific hsp70 peptides recognizedby the patients.

In one embodiment, the pharmaceutical composition is intended for theprevention or treatment of an autoimmune disease, preferably Type 1Diabetes, Systemic Lupus Erithematosus, Multiple Sclerosis or RheumatoidArthritis, and more preferably Type 1 Diabetes. Type 1 Diabetes has alsobeen known as Insulin-Dependent Diabetes Mellitus (IDDM).

Another aspect of the present invention relates to a method fordiagnosing an autoimmune disease in a patient, also at an incipientstage. Preferably, said autoimmune disease is Type 1 Diabetes, SystemicLupus Erithematosus, Multiple Sclerosis or Rheumatoid Arthritis. Morepreferably, the autoimmune disease is Type 1 Diabetes. The diagnosticmethod of the invention comprises testing a biological sample obtainedfrom a patient, preferably blood or urine sample of said patient, usinga peptide of the invention as an antigen, to detect the presence ofantibodies or T-cells which are immunologically reactive to human hsp70,whereby the presence of anti-hsp70 antibodies or of a T-cell whichimmunoreacts with hsp70 indicates an increased probability of thepresence or incipience of an autoimmune disease.

In one embodiment, the method of testing for the presence of anti-hsp70antibodies comprises a radioimmunoassay or an ELISA test.

In a second embodiment of said method of diagnosis, the patient istested for the presence of a T-cell which immunoreacts with hsp70,comprising the steps of: (a) preparing a mononuclear cell fractioncontaining T-cells from a blood sample obtained from said patient; (b)adding to said mononuclear cell fraction an antigen selected from thepeptides of the invention; (c) incubating said cell fraction in thepresence of said antigen for a suitable period of time and undersuitable culture conditions; (d) adding a labeled nucleotide to theincubated cell culture of (c) at a suitable time before the end of saidincubation period to provide for the incorporation of said labelednucleotide into the DNA of proliferating T-cells; and (e) determiningthe amount of proliferating T-cells by analysis of the amount of labelednucleotide incorporated into said T-cells by suitable means.

The diagnostic differentiation of Type 1 diabetes from Type 2 diabetesis of major importance. Type 2 diabetes is not an autoimmune disease,and is usually treated by the oral administration of insulin.Misdiagnosis of Type 1 for Type 2 diabetes, since the treatment designedfor slow-release of insulin could worsen the condition and lead toshock, and in some case, particularly in children, could belife-threatening. The present diagnostic method affords a reliablediagnosis of the Type 1 diabetes, and thus avoiding any risks which mayresult from mis-diagnosis.

The invention also provides a kit for the diagnosis of an autoimmunedisease, by testing reactivity to hsp70 antibodies in patients andsuspected patients. Preferably, the autoimmune disease to be diagnosedis Type 1 Diabetes, Systemic Lupus Erithematosus, Multiple Sclerosis orRheumatoid Arthritis. More preferably, said autoimmune disease is Type 1Diabetes.

In one embodiment, said kit provides means for conducting a test for thepresence of anti-hsp70 antibodies, and comprises the followingcomponents: (i) at least one antigen selected from the peptides of theinvention; and (ii) means for detecting said anti-hsp70 antibodies, forexample a labeled antibody capable of recognizing the non-variableregion of the anti-hsp70 antibodies.

In a second embodiment, the kit provides means for conducting a test forthe presence of a T-cell which immunoreacts with hsp70. In thisembodiment, the kit comprises the following components: (i) at least oneantigen selected from the peptides of the invention; (ii) a suitablemedium for culture of lymphocytes (T-cells); and (iii) means fordetecting T-cell proliferation, e.g. a labeled nucleotide for a T-cellproliferation test.

Lastly, the present invention provides a method of modulating anautoimmune response, in a patient in need of such treatment, whereinsaid method comprises administering to said patient a peptide selectedfrom the peptides of the invention, the full-length hsp70 or its activederivatives.

It is to be understood that as a method of modulating it is meant amethod for treatment or prevention of the autoimmune disease, which canalso be described as a method of vaccination for said disease.

In one embodiment, said peptide should be administered in a medicallyeffective amount, at least once, preferably soon after diagnosis. Thepeptide may also be administered another two times, preferably at oneand six months after the first administration, to provide a booster forthe patient.

The treatment is known to be effective when the patient in treatmentpresents, for example, the capacity to maintain his/her ability toproduce the insulin C-peptide. Alternatively, the treatment is known tobe effective when the T-cell response of the patient in treatment, inresponse to hsp70, shows increased interleukin 4, interleukin 10 andinterleukin 13 production (Th2 cytokines), for example. The productionof other cytokines, like interferon γ and IL2 (Th1 cytokines), can alsobe evaluated in T-cells of patients following treatment. The modulationof cytokine profile should reflect a shift from a pro-inflammatoryT-helper-1 (Th1) response to an anti-inflammatory T-helper-2 (Th2)response, triggered by the hsp70 peptides.

The peptide may be administered to a patient diagnosed with anautoimmune disease, preferably Type 1 Diabetes, Systemic LupusErithematosus, Multiple Sclerosis or Rheumatoid Arthritis. Morepreferably, said autoimmune disease is Type 1 Diabetes. For prevention,the treatment may be given to individuals with a genetic predispositionto one autoimmune disease, for example for individuals who had a familymember diagnosed with the same disease.

It is not known why autoimmunity to hsp60, glutamic acid decarboxylase,insulin secretory-granule 38 kDa protein (38 kDa), and other antigensnot exclusively expressed in the pancreatic islets should be associatedwith the autoimmune process leading to Type 1 Diabetes in humans andmice [Honeyman and Harrison (1993) id ibid.; Roep, B. (1996) id ibid.;Rudy, G. et al. (1995) Molecular Medicine, 1:625-633]. Autoimmunity tohsp70 is likely to have a functional role in the Type 1 Diabetes processduring childhood. Here the inventors have shown that an immunoreactionto hsp70 can be a useful marker for the detection of Type 1 Diabetes,particularly in children. Potentially the hsp70 protein or itsderivatives could be used in a vaccine to treat such children, as wellas adults (Table 1D). Such hsp70 vaccine could also be used to treatindividuals carrying other autoimmune diseases, like Type 1 Diabetes,Systemic Lupus Erithematosus, Multiple Sclerosis or RheumatoidArthritis. More preferably, said autoimmune disease is Type 1 Diabetes.

Intact hsp70 and hsp70 major peptides may be tested as vaccines toprevent the progression of diabetes in NOD mice.

The hsp70 peptides of the present invention can be administered in avariety of ways to modulate the immune response of an individual (e.g.,a human, other mammal or other vertebrate). In another embodiment, theprotein or peptide is administered as a vaccine which is comprised of atleast one hsp70 peptide of the invention, or a portion of it, which isof sufficient size to stimulate the desired immune response.

Alternatively, T-cell vaccination could be used. For that purpose, hsp70specific, auto-reactive T-cells are isolated from the patient in need ofsaid treatment, and activated in vitro with hsp70 protein or peptides.The responding T-cells are then selected and isolated, expanded, andfinally attenuated. The attenuated T-cells are then administered to thepatient, as the T-cell vaccine.

Therefore, the method of the present invention can also be used tomodify or modulate an individual's response to his or her own cells, inan autoimmune disease. As shown by the inventors, hsp70 protein isinvolved in Type 1 Diabetes, an autoimmune disease. It is, thus,possible to turn down an individual's immune response, resulting in theindividual becoming more tolerant to the protein. It is possible toselectively inhibit or interfere with the ability of immune cells whichnormally interact with such proteins to do so.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES Experimental Procedures

Subjects

(a) Type 1 Diabetes patients: 25 children (mean age 10.2±4.2 years),consecutively admitted to the Department of Pediatric Endocrinology ofthe Hadassah University Hospital (Hebrew University of Jerusalem,Israel), were enrolled in the study, with informed consent obtained fromthe parents. The mean time elapsed from the time of diagnosis was 3.8weeks (range 1-8 weeks). The criteria for Type 1 Diabetes diagnosiswere: classical clinical symptoms, including a recent history ofpolyurea, polydipsia, weight loss with or without associated severeketoacidosis, ketonuria and hyperglycemia (glucose≧200 mg/dl or 11.1mM/l)

b) Type 2 Diabetes patients: Eleven adult patients (mean age 63±6.6years) diagnosed as Type 2 Diabetes/NIDDM patients at the Department ofEndocrinology, Hadassah University Hospital (Hebrew University ofJerusalem, Israel), provided blood samples with informed consent. In anattempt to control the possible effects of disease severity and insulintherapy, the Type 2 Diabetes patients were all in need of insulin tomanage their hyperglycemia. The mean duration of disease of this groupwas 20.1±7.7 years. Mean time of insulin treatment was 6.6±4.9 years.

c) Healthy Blood Donors: Samples of surplus blood to be used as controlwere obtained from the Blood Bank (Tel HaShomer Hospital, Tel Aviv,Israel) from 25 healthy adult donors. It was necessary to use most ofthe control samples from healthy adults because drawing blood fromhealthy children is unacceptable practice, unless in the below-specifiedcircumstances.

d) Blood sera from healthy children: Blood sera from 10 normoglycemicchildren (mean age 6.5±4.5 years) were obtained, after parentalpermission, from children admitted to the Emergency Room of the HadassahUniversity Hospital (Jerusalem, Israel) for treatment of acute medicalconditions.

e) Pediatric T-cell donors: Samples of surplus blood from three childrenwas also obtained, with parental permission, from which T cells wereisolated. These children (two females, one male; ages 3, 7 and 10 yearsold) were not Type 1 diabetes patients, and were admitted to theDepartment of Pediatrics, Hadassah University Hospital, HebrewUniversity of Jerusalem (Jerusalem, Israel).

HsD70 Peptides

Hsp70 peptides were prepared in the Biological Services Laboratory ofthe Weizmann Institute of Science (Rehovot, Israel), using an automatedAbimed Synthesizer (Model MAF422, Langenfeld, Germany). The peptideswere purified by reverse phase HPLC and their compositions wereconfirmed by amino acid analysis. The peptides were provided lyophilizedand stored at −20° C. Prior to use, the peptides were dissolved in PBSto a concentration of 5-20 μg/ml, and the remaining of the dissolvedpeptides was stored at −20° C. for further usage. Except for p43, allthe hsp70 peptides were 20 amino acids in length, with 5 overlappingamino acids in each side. The amino acid sequences of all of thepeptides used herein are shown in Table 2.

T-cell Proliferation Assay

For T-cell proliferation assays, 30-50 ml of peripheral blood was usedas described [Abulafia-Lapid et al. (1999) id ibid.]. Anti-coagulationwas achieved with [10 IU/ml heparin]. Peripheral blood mononuclear cells(PBMC) were isolated by Ficoll Paque (Pharmacia Biotech, Uppsala Sweden)density centrifugation. The cells were washed with RPMI culture media(Biological Industries, Kibbutz Beit Haemek, Israel), supplemented with1% Sodium-Pyruvate, 1% L-Glutamine (200 mM), 1% Penicillin/Streptomycin(10,000 U/ml/10,000 mg/ml) and 2% Hepes (1 M, pH 7.3) (all fromBiological Industries, Beit Haemek, Israel). PBMC were plated intriplicate or in quadruplicate in 96-well round-bottom micro plates(Falcon, Lincoln Park, N.J., USA) at a cell concentration of 2×10⁵ cellsper well in 100 μl RPMI media, with or without the following testantigens: PHA (Murex Diagnostic Ltd. England) 0.3 μg/ml; Tetanus Toxoid(Connaught Lab. Inc, Penn., USA) 5 μg/ml; Candida Albicans 20 μg/ml,recombinant human hsp60, hsp70 and hsp90 (StressGen, Canada), 2-5 μg/ml;and hsp70 peptides, 5-20 μg/ml (synthesized at the Biological ServicesLaboratory of the Weizmann Institute of Science, Rehovot, Israel, usingan automated ABIMED synthesizer AMA422, Langenfeld, Germany). Allproliferation assays, including the epitope mapping, were performed inRPMI medium supplemented with 10% autologous serum, and incubated at 37°C. in a 5% CO₂ humidified incubator for 7 days. On day 6, the cells werelabeled with 1 μCi/well of ³H-Thymidine. On day 7, the radioactivity wascounted using a beta-counter (Packard model 2000).

Proliferation was represented as stimulation index (S.I.), obtained fromthe ratio between the mean value of proliferation (in cpm) with antigenand the mean value of proliferation without antigen (in cpm). S.I.values were considered positive when greater than or equal to 2.

Determination of Auto-Antibodies

All of the serum samples that were collected were stored in aliquotes at−20° C. and thawed before each assay. Total human IgG anti-hsp60,anti-hsp70, and anti-hsp90, as well as anti-tetanus toxoid proteins weredetected by the antibody capture-type enzyme immunoassay ELISA [Maggio,M. T. (1981) Enzyme Immunoassay, CRC Press, Boca Raton, USA]. 96-wellmicro titer plates (Dynatech) were coated with the protein according tothe antibody being tested. Serum samples were distributed at dilutionsin the range of 1:50 to 1:70 into the pre-coated wells for the proteindetection assay. Thus, any IgG anti-hsp60, anti-hsp70, anti-hsp90, oranti-tetanus toxoid present should be bound by the immobilized peptideor protein. After washing off unbound material, a mouse monoclonalanti-human IgG antibody conjugated to alkaline phosphatase (AP) wasadded to the wells. After an additional washing step to remove anyunbound anti-enzyme reagent, the AP substrate p-nitrophenyl phosphate(pNPP) solution was added. The intensity of the color developed wasmeasured using an Anthos htll ELISA reader at λ=405 nm. The amount ofantibody detected was represented as Units of Optical Density at λ=405nm. Patients and control subjects were assayed in the same experiment.

Statistical Analysis

The InStat 2.01 computer program was used for the statistical analysis.The results obtained from the samples of healthy individuals were usedto determine the cutoff for each assay, which was established as themean plus two standard deviations (SDs). Samples that displayed valuesabove this calculated cutoff were considered positive. The results werepresented as the percentage of positive from the total tested group.

In addition, p values were approximated using the two-tailed Fisher'sexact test.

Example 1

T-cell Responses to hsp70

T-cell responses to intact hsp60, hsp70, and hsp90 proteins, and to therecall antigens tetanus toxoid and Candida albicans were comparedbetween the Type 1 Diabetes children (Table 1A), the healthy blood donorsubjects (Table 1B) and the Type 2 Diabetes patients (Table 1C).

Table 1: T-cell proliferative response to hsp60, hsp70, or hsp90 TABLE1A Type 1 Diabetes children Age Subject (years) Weeks* T.T. Candidahsp60 hsp70 hsp90 P1 16 3 5.4 6.5 1.3 1.3 1.2 P2 5 5 24.0 19.2 3.6 2.01.0 P3 13 3 34 38 2.5 1.7 1 P4 9 1 13.8 7.3 1.0 4.8 1.4 P5 14 3 40.635.2 6.0 7.3 3.8 P6 14 4 6.7 3.9 3.3 5.6 1.0 P7 1.8 3 72.8 23.5 5.5 3.31.1 P8 9.5 4 10.9 13.0 1.4 2.1 1.6 P9 13 3 14.2 25.4 6.1 7.8 1.0 P10 5 339.3 4.3 2.1 14.3 1.5 P11 9 3 4.5 3.6 3.7 8.0 3.0 P12 16 3 7.1 1.7 1.91.0 1.0 P13 18 3 25.0 15.3 13.4 8.9 1.7 P14 10 3 21.4 18.5 2.4 2.3 1.6P15 6 2 9.2 8.1 2 1 2.6 P16 7.5 3 10.1 10.5 4.6 4.9 0.8 P17 9 3 4.2 6.32.4 1.5 1 P18 9 4 10.2 6.3 3.7 3.7 1 P19 6.5 3 4.5 5.3 7.1 2 1 P20 10 317.7 13 2.7 4.4 5.1 P21 16 8 nd 23 1 4.9 1.9 P22 10.5 2 5.6 7.1 3.6 4.43.7 P23 7 12 17.4 18 3 6.2 n.d. P24 9 4 5.5 9.4 4.5 4.7 1.5 P25 8 8 7.76.9 2.1 4.4 1.5 Mean ± SD 10.1 ± 3.8 3.8 ± 2.3 17.2 ± 15.9 13.2 ± 9.63.6 ± 2.6 4.5 ± 3.1 1.7 ± 1.1 (p = 0.035) (n.s.) (p < 0.0001) (p <0.0001) (n.s.)*Weeks since diagnosis.nd = not done.n.s. = not statistically significant.

TABLE 1B Healthy subjects Sample Tetanus No. Toxoid Candida hsp60 hsp70hsp90 C1 1 16 1.9 2.1 2.5 C2 50 26 2.5 1.5 2.3 C3 13.8 18.3 2.1 1 1.8 C42.9 2.5 1 1 1 C5 8.7 6.5 2.6 1.3 4 C6 3 5 1.3 1.4 1.3 C7 15.7 8.5 1 1.11 C8 3.2 10.5 1.4 1.5 2.2 C9 10.6 12.3 2 1 1.8 C10 4.3 7.1 1.75 1 1.4C11 11.3 17.8 2.9 1 1 C12 3.5 3.6 2.9 1.5 1 C13 5.5 11.1 2.3 1.7 1.9 C149 4.5 1 1 1 C15 19.3 13.9 1.3 1.6 1 C16 3 6 1 1 1.5 C17 6.6 8.8 2.5 1.51.2 C18 4.3 8.9 3.5 3.2 2.4 C19 2.5 5.1 1.3 1.3 2.4 C20 2.4 4.6 1 1.5 2C21 3.4 7.3 1.9 1 1 C22 10.7 25 1 3.1 1.1 C23 2.9 2.5 1 1 1 C24 2.7 3.71.5 1.9 1 C25 11.1 77 11 22 11 C26* 7.3 16 1.25 1.1 1 C27* 3.5 3.6 1 1 1C28* 2.2 2.1 1 1 1 Mean ± SD 8.01 ± 9.3 9.4 ± 6.4 1.7 ± 0.72 1.44 ± 0.61.52 ± 0.7C26*-C28*: Pediatric T-cell donors

TABLE 1C Type 2 Diabetes Sample Age Tetanus Candi- No. (years) Toxoid dahsp60 hsp70 hsp90 N1 75 2.7 5.1 1.1 1.3 1.2 N2 63 10.1 nd 1.3 1 1 N3 658.1 nd 2.6 1 1 N4 60 Nd 45 1.5 1.2 1.5 N5 63 1 1 1 1.1 1.2 N6 60 Nd 5.22.4 2.1 1.8 N7 72 3.7 2.5 1 1 1 N8 60 3.6 5.2 1.2 1.1 1.3 N9 50 2.5 nd3.3 3.9 1 N10 66 3 nd 1.7 4.6 5.7 N11 60 4.6 2.5 1.8 1.45 1 Mean ± 63 ±4.4 ± 9.5 ± 1.7 ± 0.75 1.8 ± 1.26 1.6 ± 1.38 SD 6.6 2.9 15.7nd = not done.

TABLE 1D T-cell responses of Type 1 Diabetes adult patients Time sincediagnosis Tetanus Patient Age Gender (weeks) toxoid Candida hsp60 hsp70hsp90 AP1 25 M 12 1 — 1 1.70 1.13 AP2 20.1 F 19 7.8 nd 1.2 2.09 1.22 AP320 M 10 8.4 — 2.1 3.20 1.39 AP4 18 M 8 3.8 nd 1.2 2.8 1.20 AP5 19 F 165.9 nd 1 2.00 — AP6 25 M 12 3.2 1.5 1.1 2.1 1.40 AP7 22 M 12 70 8.3 14.3 2.20 Mean ± SD 21.3 ± 2.59 12.71 ± 3.41 14.3 ± 22.86 4.9 ± 3.4 1.22± 0.36 2.6 ± 0.84 1.4 ± 0.3

Type 1 Diabetes patients were identified as P1 to P25, healthy subjects(control) as C1-C28, and Type 2 Diabetes patients as N1-N11. Threepediatric controls are C26-C28. Adult patients are AP1-AP1 (Table 1D).

T-cells were isolated from 25 children newly diagnosed with Type 1Diabetes (Table 1A), 28 healthy subjects (Table 1B), and 11 Type 2Diabetes patients (Table 1C). The subjects were tested for theirproliferative responses to hsp60, hsp70, and hsp90 and also to recallantigen Tetanus Toxoid and Candida albicans. The responses, shown asStimulation Index (S.I.), represent the ratio of the mean T-cellresponse with antigen to the T-cell response without antigen. A value ofS.I. equal or greater than 2 was considered positive. T-cell responsewas evidenced by cell proliferation measured in cpm. Statisticalanalysis was performed using the InState 2.01 computer program, andp-values were approximated by the Krusal-Wallis nonparametric ANOVAtest.

Surprisingly, T-cell responses of Type 1 Diabetes children to hsp70(Table 1A) were significantly higher than those of the other two groups.There were significantly more responding Type 1 Diabetes children (20 of25; 85%) than healthy blood donors (4 out of 28; 14%) (p=0.0006) or Type2 Diabetes patients (3 out of 11; 27%) (p=0.0006). The degree ofresponsiveness to hsp70 was also higher in the Type 1 Diabetes group(mean S.I.=4.5±3.1) when compared to that of the Type 2 Diabetes group(mean S.I.=1.8±1.3) or to the healthy blood donors (mean S.I.=1.4±0.6;p<0.0001). Thus, recently diagnosed Type 1 Diabetes children had anenhanced T-cell proliferative response to hsp70. Although 20 of the 25Type 1 Diabetes children responded to hsp60, this response was lower(mean of S.I.=3.6±2.6) than the response to hsp70 (mean ofS.I.=4.5±3.1). Interestingly, three Type 1 Diabetes patients (P4, P8 andP21) responded exclusively to hsp70 and not to hsp60 (or hsp90). It isimportant to highlight that the difference between the responses of Type1 Diabetes patients and healthy individuals to hsp70 (SI_(IDDM)=40±3.1versus SI_(healthy)=1.45±0.6, or 85% versus 13%, respectively) is higherthan the difference of their responses to hsp60 (SI_(IDDM)=3.6±2.6versus SI_(healthy)=1.79±0.74, or 85% versus 39%, respectively). In sum,these results demonstrate that the T-cell response to hsp70 is a moredistinctive parameter for the diagnosis and treatment of Type 1Diabetes.

In contrast, 5 out of 24 (21%) Type 1 Diabetes children tested respondedto hsp90 (mean S.I.=1.7±1.2). In the other two groups, there were 7 outof 28 healthy subjects (25%) (mean S.I.=1.52±0.7) and 1 out of 11 (9%)Type 2 Diabetes subjects (mean S.I.=1.6±1.4) that responded to hsp90.

The three groups tested, shown in Tables 1A, 1B and 1C respondedsimilarly to the Candida antigen. The responses of the Type 1 Diabeteschildren (mean S.I.=17.2±16.1, Table 1A) to the tetanus toxoid werehigher than those of the healthy subjects (S.I.=9.4±6.4, p=0.035; Table1B), whereas the responses of the Type 2 Diabetes subjects were lowerthan the latter (S.I.=4.4±2.9, Table 1C). This result was expected,since the response to tetanus toxoid is usually higher in youngerpatients due to the fact that these individuals likely received abooster shot immunization more recently than older individuals.Therefore, the result of the Type 2 Diabetes group was the lowest,probably because they were older (mean age of 63±6.6) than the subjectsin the other two groups, and thus even less likely to have received atetanus toxoid booster shot in the recent past.

It is important to mention that, although the sample size is small, theresults obtained for T cell responses from C26-C28 (healthy children) tohsp60, hsp70 and hsp90 were comparable to that of C1-C25 (healthyadults).

From the results shown in Table 1D it can be seen that these adult Type1 patients, whose age range was 18-23, nicely responded to hsp70 within12 weeks from diagnosis (about 86%, mean=2.6+0.84). A response to hsp60treatment is not seen, possibly because these patients were tested aftermore than 12 weeks. This means that the response to hsp70 is longer. Noresponse to hsp90 was observed, possibly because the test was late, ascompared to children. It appears from these preliminary results thatanti-hsp70 antibodies may be better than anti-hsp60 and anti-hsp90.

Dynamics of the hsp70 and hsp60 Responses

In FIG. 1, the responsiveness of the Type 1 Diabetes children to hsp70can be compared with that to hsp60, and the magnitude of the T-cellresponses can be appreciated (FIG. 1 and Table 1A). Amongst the 20 Type1 Diabetes subjects that responded to hsp70, 17 (85%) also responded tohsp60, and from these, 12 had their hsp70 response either higher orequal to the hsp60 response (FIG. 1). It is important to note that themean S.I. value of the hsp70 response was higher than that of the hsp60response, and from those patients that responded to both hsp70 andhsp60, the majority responded better to hsp70. This strengthens theinventor's finding that hsp70 is an ideal treatment and diagnostic toolfor Type 1 Diabetes.

Example 2

Epitope Mapping of the hsp70 Peptides

In order to determine the spectrum of the hsp70 peptides that wererecognized in the Type 1 Diabetes children, T-cell proliferativeresponses to the 43 overlapping hsp70 peptides (Table 2) were assayed(FIG. 2). TABLE 2 Overlapping peptides of the human hsp70 moleculePeptide Sequence ID Number Position Sequence SEQ. ID. NO. 1 p1  1-20MAKAAAVGIDLGTTYSCVGV SEQ. ID. NO. 2 p2 16-35 SCVGVFQHGKVEIIANDQGN SEQ.ID. NO. 3 p3 31-50 NDQGNRTTPSYVAFTDTERL SEQ. ID. NO. 4 p4 46-65DTERLIGDAAKNQVALNPQN SEQ. ID. NO. 5 p5 61-80 LNPQNTVFDAKRLIGRKFGD SEQ.ID. NO. 6 p6 76-95 RKFGDPVVQSDMKHWPFQVI SEQ. ID. NO. 7 p7  91-110PFQVINDGDKPKVQVSYKGE SEQ. ID. NO. 8 p8 106-125 SYKGETKAFYPEEISSMVLT SEQ.ID. NO. 9 p9 121-140 SMVLTKMKEIAEAYLGYPVT SEQ. ID. NO. 10 p10 136-155GYPVTNAVITVPAYFNDSQR SEQ. ID. NO. 11 p11 151-170 NDSQRQATKDAGVIAGLNVLSEQ. ID. NO. 12 p12 166-185 GLNVLRIINEPTAAAIAYGL SEQ. ID. NO. 13 p13181-199 IAYGLDRTGKGERNVLIFDL SEQ. ID. NO. 14 p14 195-214LIFDLGGGTFDVSILTIDDG SEQ. ID. NO. 15 p15 210-229 TIDDGIFEVKATAGDTHLGGSEQ. ID. NO. 16 p16 225-244 THLGGEDFDNRLVNHFVEEF SEQ. ID. NO. 17 p17240-259 FVEEFKRKHKKDISQNKRAV SEQ. ID. NO. 18 p18 255-275NKRAVRRLRTACERAKRTLS SEQ. ID. NO. 19 p19 271-290 KRTLSSSTQASLEIDSLFEGSEQ. ID. NO. 20 p20 286-305 SLFEGIDFYTSITRARFEEL SEQ. ID. NO. 21 p21301-320 RFEELCSDLFRSTLEPVEKA SEQ. ID. NO. 22 p22 316-335PVEKALRDAKLDKAQIHDLV SEQ. ID. NO. 23 p23 331-350 IHDLVLVGGSTRIPKVQKLLSEQ. ID. NO. 24 p24 346-365 VQKLLQDFFNGRDLNKSINP SEQ. ID. NO. 25 p25361-380 KSINPDEAVGYGAAVQAAIL SEQ. ID. NO. 26 p26 376-395QAAILMGDKSENVQDLLLLD SEQ. ID. NO. 27 p27 391-410 LLLLDVAPLSLGLETAGGVMSEQ. ID. NO. 28 p28 406-425 AGGVMTALIKRNSTIPTKQT SEQ. ID. NO. 29 p29421-440 PTKQTQIFTTYSDNQPGVLI SEQ. ID. NO. 30 p30 436-455PGVLIQVYEGERAMTKDNNL SEQ. ID. NO. 31 p31 451-470 KDNNLLGRFELSGIPPAPGVSEQ. ID. NO. 32 P32 466-485 PAPGVPQIEVTFDIDANGIL SEQ. ID. NO. 33 p33481-500 ANGILNVTATDKSTGKANKI SEQ. ID. NO. 34 p34 496-515KANKITITNDKGRLSKEEIE SEQ. ID. NO. 35 p35 511-530 KEEIERMVQEAEKYKAEDEVSEQ. ID. NO. 36 p36 526-545 AEDEVQRERVSAKNALESYA SEQ. ID. NO. 37 p37541-560 LESYAFNMKSAVEDEGLKGK SEQ. ID. NO. 38 p38 556-575GLKGKISEADKKKVLDKCQE SEQ. ID. NO. 39 p39 571-590 DKCQEVISWLDANTLAEKDESEQ. ID. NO. 40 p40 586-605 AEKDEFEHKRKELEQVCNPI SEQ. ID. NO. 41 p41601-620 VCNPIISGLYQGAGGPGPGG SEQ. ID. NO. 42 p42 616-635PGPGGFGAQGPKGGSGSGPT SEQ. ID. NO. 43 p43 631-640 GSGPTIEEVD

In FIG. 2, seven Type 1 Diabetes patients (P3, P16, P18, P20, P21, P23and P24 from Table 1A) were tested for their responsiveness to the 43hsp70 peptides (Table 2). Reactivity was measured as T-cell response toeach of the peptides in a proliferation assay as described above.Peptides were considered immunogenic when at least 3 out of the 7patients tested had a positive response. As before, a S.I. value of 2and greater was considered positive. Amongst the seven subject samplestested, there was reactivity to nine of the 43 peptides. More precisely,the nine peptides to which there was reactivity were: p1, p12, p15, p16,p19, p27, p29, p34 and p35 (FIG. 2). Patients P24 and P23 reacted to sixand seven out of these nine peptides, respectively. Because six out ofseven of the Type 1 Diabetes subjects tested responded to peptide p27(residues 391-410, SEQ. ID. NO.27), five responded to peptide p35(residues 511-530, SEQ. ID. NO.35), and five (P21, P23 and P24, see FIG.2A, and two Type 1 Diabetes adult patients, data not shown) responded top1 (residues 1-20, SEQ. ID. NO.1), these three peptides were consideredthe major antigenic peptides in the hsp70 protein. In conclusion,multiple hsp70 peptides appear to be recognized by the Type 1 Diabetespopulation, amongst which, three of them (p1, p27 and p35) seem to beits hallmark and harbor major antigenic sites.

Example 3

Auto-Antibodies to hsp60, hsp70, and hsp90

Levels of IgG antibodies to hsp60, hsp70, and hsp90 were measured in thesera of 20 Type 1 Diabetes children (P1-P5, P7-P21; Table 3A) and twocontrol groups, 10 normoglycemic children (Table 3B) and 15 healthyadult blood donors (Table 3C). The level of the antibodies was scored aspositive when it was greater than the cut-off level (cut-off levels wereestablished based on the mean value of antibody levels from thenormoglycemic children plus two standard deviations). TABLE 3A IgGantibodies to hsp60, hsp70 and hsp90 in Type 1 diabetes childrenDiabetes duration Subject Age/Sex (Weeks*) Hsp60 Hsp70 Hsp90 P1  16/F 31.04 0.53 0.66 P2   5/F 5 0.86 2 0.68 P3  13/F 3 0.45 0.28 0.45 P4   9/F1 0.67 0.37 0.45 P5  14/M 3 0.77 0.7 0.44 P7 1.8/M 3 0.29 0.26 0.35 P89.5/M 4 0.69 0.34 0.59 P9  13/M 3 0.57 0.3 0.37 P10   5/F 3 0.46 0.260.45 P11   9/F 3 0.34 0.25 0.39 P12  16/M 3 0.46 0.27 0.39 P13  18/M 31.25 0.75 0.82 P14  10/M 3 0.51 0.29 0.45 P15   6/F 2 0.72 0.33 0.27 P167.5/F 3 0.76 1.22 0.6 P17   9/F 3 0.54 0.28 0.43 P18   9/F 4 0.49 0.290.37 P19 6.5/F 3 0.35 0.23 0.38 P20  10/M 3 0.39 0.88 0.33 P21  16/F 80.73 0.44 0.56 Mean ± SD 10.1 ± 4.1 3.3 ± 1.2 0.6 ± 0.5 ± 0.41 0.47 ±0.13 0.23 Cut-off 0.67 0.53 0.64 Positive 45% 30% 15%*weeks since diagnosis

TABLE 3B IgG antibodies to hsp60, hsp70 and hsp90 in pediatric controlserum donors Subject Age/Sex Hsp60 Hsp70 Hsp90 C1 7/M 0.61 0.41 0.41 C23/M 0.39 0.34 0.54 C3 12/M  0.35 0.45 0.47 C4 13/M  0.28 0.26 0.41 C510/M  0.47 0.47 0.56 C6 0.5/M   0.39 0.35 0.3 C7 3/M 0.43 0.27 0.3 C82/M 0.5 0.37 0.55 C9 4/F 0.4 0.17 0.28 C10 10/M  0.67 0.44 0.5 Mean ± SD6.45 ± 4.3 0.45 ± 0.11 0.35 ± 0.09 0.43 ± 0.1 Cut-off 0.67 0.53 0.64Positive 10% 0 0

TABLE 3C IgG antibodies to hsp60, hsp70 and hsp90 in healthy adult blooddonors Subject Hsp60 Hsp70 Hsp90 AC1 0.53 0.30 0.42 AC2 0.57 0.35 0.45AC3 0.76 0.70 0.41 AC4 0.75 0.50 0.74 AC5 0.57 0.35 0.47 AC6 0.38 0.260.31 AC7 0.52 0.44 0.49 AC8 0.62 0.41 0.45 AC9 0.63 0.46 0.49 AC10 0.890.52 0.61 AC11 0.58 0.38 0.44 AC12 0.52 0.35 0.3 AC13 0.59 0.5 0.47 AC140.64 0.52 0.40 AC15 0.67 0.41 0.49 Mean ± SD 0.61 ± 0.12 0.43 ± 0.1 0.46± 0.1 Cut-off 0.85 0.64 0.67 Positive 6.6% 6.6% 6.6%

Of the Type 1 Diabetes children, 45% (9 out of 20) were positive tohsp60, 30% (6 out of 20) were positive to hsp70, and 15% (3 out of 20)were positive to hsp90 (FIG. 3). Out of the nine Type 1 Diabeteschildren positive to hsp60, five were also positive to hsp70. Incontrast, of the 10 healthy children, only one (10%) was positive tohsp60, and none were positive to hsp70 or to hsp90 (FIG. 3). Among thehealthy adult blood donors, only 6% were positive to hsp60, hsp70 andhsp90. It is remarkable that although only 30% of the Type 1 Diabeteschildren were positive to hsp70, none of the healthy children weresero-positive to hsp70. This result shows that being sero-positive tohsp70 is one good indicator of Type 1 Diabetes in children. Antibodiesto tetanus toxoid were measured as control, and were comparably high inboth Type 1 Diabetes and control groups.

There was no correlation between the IgG antibody levels and themagnitude of the T-cell response to these proteins.

Example 4

Hsp70 Vaccination

Groups of 10 female NOD mice are treated at age 4-6 weeks with 100 μg ofhsp70 peptides p1, p27 and p35, and IFA (incomplete freund's adjuvant)as a control group. The peptides are emulsified in oil (IFA). The NODmice are injected subcutaneously. In order to examine development ofhyperglycemia, blood glucose level may be monitored every two weeksusing glucose analyzer.

1. A peptide selected from the group consisting of the peptides denotedby SEQ. ID. NO.1, SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID.NO.5, SEQ. ID. NO.6, SEQ. ID. NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ.ID. NO.10, SEQ. ID. NO.11, SEQ. ID. NO.12, SEQ. ID. NO.13, SEQ. ID.NO.14, SEQ. ID. NO.15, SEQ. ID. NO.16, SEQ. ID. NO.17, SEQ. ID. NO.18,SEQ. ID. NO.19, SEQ. ID. NO.20, SEQ. ID. NO.21, SEQ. ID. NO.22, SEQ. ID.NO.23, SEQ. ID. NO.24, SEQ. ID. NO.25, SEQ. ID. NO.26, SEQ. ID. NO.27,SEQ. ID. NO.28, SEQ. ID. NO.29, SEQ. ID. NO.30, SEQ. ID. NO.31, SEQ. ID.NO.32, SEQ. ID. NO.33, SEQ. ID. NO.34, SEQ. ID. NO.35, SEQ. ID. NO.36,SEQ. ID. NO.37, SEQ. ID. NO.38, SEQ. ID. NO.39, SEQ. ID. NO.40, SEQ. ID.NO.41, SEQ. ID. NO.42 and SEQ. ID. NO.43, and salts, analogues andfunctional derivatives thereof.
 2. A peptide according to claim 1,wherein said functional derivatives consist of chemical modifications toamino acid side chains and/or the carboxyl and/or amino moieties of saidpeptides.
 3. A peptide according to claim 1, selected from the groupconsisting of the peptides SEQ. ID. NO.1, SEQ. ID. NO.12, SEQ. ID.NO.15, SEQ. ID. NO.16, SEQ. ID. NO.19, SEQ. ID. NO.27, SEQ. ID. NO.29,SEQ. ID. NO.34 and SEQ. ID. NO.35.
 4. A peptide according to claim 3,selected from the group consisting of peptides SEQ. ID. NO.1, SEQ. ID.NO.27 and SEQ. ID. NO.35.
 5. A pharmaceutical composition comprising atleast one peptide according to claim 1 and optionally comprising apharmaceutically acceptable carrier.
 6. A pharmaceutical compositioncomprising at least one peptide according to claim 3 and optionallycomprising a pharmaceutically acceptable carrier.
 7. A pharmaceuticalcomposition comprising at least one peptide according to claim 4 andoptionally comprising a pharmaceutically acceptable carrier.
 8. Thepharmaceutical composition according to claim 6 for the prevention ortreatment of an autoimmune disease.
 9. A pharmaceutical compositionaccording to claim 8, wherein said autoimmune disease is Type 1Diabetes, Systemic Lupus Erithematosus, Multiple Sclerosis or RheumatoidArthritis.
 10. A pharmaceutical composition according to claim 9,wherein said autoimmune disease is Type 1 Diabetes.
 11. A method fordiagnosing the occurrence or incipience of an autoimmune disease in apatient by use of a peptide as defined in claim
 1. 12. The methodaccording to claim 11, wherein said autoimmune disease is Type 1Diabetes, Systemic Lupus Erithematosus, Multiple Sclerosis or RheumatoidArthritis.
 13. The method according to claim 12, wherein said autoimmunedisease is Type 1 Diabetes.
 14. The method according to claim 11,wherein said method comprises testing a blood or urine sample of saidpatient for the presence of antibodies or T-cells which areimmunologically reactive to human hsp70 by contacting said sample withat least one peptide as defined in claim 3 or 4, and detecting animmunoreaction between said sample and said peptide, wherein thepresence of such immunoreaction indicates the presence of anti-hsp70antibodies or of a T-cell, indicating an increased probability of thepresence or incipience of an autoimmune disease.
 15. The methodaccording to claim 14, wherein said patient is tested for the presenceof anti-hsp70 antibodies.
 16. The method according to claim 14, whereinsaid immunoreaction is detected by radioimmunoassay.
 17. The methodaccording to claim 14, wherein said immunoreaction is detected by anELISA test.
 18. The method according to claim 14, wherein said patientis tested for the presence of a T-cell which immunoreacts with hsp70.19. The method, wherein said patient is tested for the presence of aT-cell which immunoreacts with hsp70, and wherein said method comprisesthe steps of: (a) preparing a mononuclear cell fraction containingT-cells from a blood sample obtained from said patient; (b) adding tosaid mononuclear cell fraction at least one antigen selected from thepeptides defined in claim 3; (c) incubating said cell fraction in thepresence of said antigen for a suitable period of time and undersuitable culture conditions; (d) adding a labeled nucleotide to theincubated cell culture of (c) at a suitable time before the end of saidincubation period to provide for the incorporation of said labelednucleotide into the DNA of proliferating T-cells; and (e) determining bysuitable means the amount of proliferating T-cells by analysis of theamount of labeled nucleotide incorporated into said T-cells.
 20. A kitfor the diagnosis of an autoimmune disease by testing for the presenceof anti-hsp70 antibodies, wherein said kit comprises the followingcomponents: (f) At least one antigen selected from peptides as definedin claim 3; and (g) a tagged antibody capable of recognizing thenon-variable region of said anti-hsp70 antibodies.
 21. The kit accordingto claim 20, wherein said autoimmune disease is Type 1 Diabetes,Systemic Lupus Erithematosus, Multiple Sclerosis or RheumatoidArthritis.
 22. The kit according to claim 21, wherein said autoimmunedisease is Type 1 Diabetes.
 23. A kit for the diagnosis of an autoimmunedisease by testing for the presence of a T-cell which immunoreacts withhsp70, wherein said kit comprises the following components: (h) at leastone antigen selected from the peptides as defined in claim 3; (i) asuitable medium for culture of lymphocytes (T-cells); and (j) a labelednucleotide for a T-cell proliferation test.
 24. The kit according toclaim 23, wherein said autoimmune disease is Type 1 Diabetes, SystemicLupus Erithematosus, Multiple Sclerosis or Rheumatoid Arthritis.
 25. Thekit according to claim 24, wherein said autoimmune disease is Type 1Diabetes.
 26. A method of modulating an immune response, in a patient inneed of such treatment, wherein said method comprises administering tosaid patient a peptide selected from the peptides as defined in claim 3.27. The method according to claim 26, wherein said method comprisesadministering the peptide in a medically effective amount, at leastonce, to the patient in need of such treatment.